Pesticidal and antiparasitic compositions

ABSTRACT

This invention relates to pesticide and antiparasitic compositions for the control of pests, diseases and parasites attacking plants and animals. The compositions include, at least one chitinolytic agent or a chitinolytic activity-inducing agent, and sulfide or a sulfide-producing agent from microorganisms or chemical compounds, wherein the chitinolytic agent or the chitinolytic activity-inducing agent and sulfur or a sulfur-producing agent obtaining from microorganisms or chemical compounds are concurrently applied at a range significantly lower than any of the above-mentioned compounds, when they are individually to attain effective control.

[0001] This invention comprises several synergistic compositions, of thepesticide and antiparasitic kind, useful for the control of parasiticphytonematodes and zoonematodes, some diseases (fungal and bacterial),and the control of parasitic trematodes (Fasciola hepatica).

PRIOR ART

[0002] Nematodes are blamed for causing the greatest damages toagriculture in tropical, subtropical and temperate regions worldwide(Nickle W. R. (Editor). 1991. Manual of Agricultural Nematology, MarcelDekker, Inc., New York, N.Y. Pub. 1035 pp). Plantain alone has about 20%nematode-related losses of world production, representing $178 millionseach year (Sasser J. N. and Freckman D. W. 1987. A world perspective onnematology: the role of the society. Vistas on nematology: acommemoration of the twenty-fifth anniversary of the Society ofNematologists/edited by Joseph A. Veech and Donald W. Dickson. p. 7-14).Plantain and banana plantations are significantly affected by Radopholussimilis.

[0003] Meloidogyne spp is the most important plant parasitic nematode,for its activity causes losses between 11% and 25% of crops in almostall the tropical regions (Sasser J. N. 1979. Root-knot nematodes. Ed. F.Lamberti & C. E. Taylor, Academic Press, London, p 359). Consequently,there is a great need to control those parasites that were foughtagainst with chemical nematicides in the past. Such compounds can behighly effective; however, many of them pose a great danger on theenvironment. In some cases the regulating authorities have limited theamount or frequency, or both in the use of such compounds, thuscompromising their nematicidal effectiveness.

[0004] Nematode control still falls short. The use of chemicalnematicides is restricted each day more and more, because they havehighly toxic and widespread action compounds. As a result, efforts havebeen made to identify the effective means to eliminate the damage causedby nematodes, in favor of reducing the use of chemical pesticides. Oneof the approaches is the use, of biological ones with specific mode ofactions and relatively safer toxicological profiles, instead of chemicalnematicides. Some of the alternative nematicides include ABG-9008, aMyrothecium verrucaria fungus metabolite and a combination ofavermectines (or related compounds, like milbecines) with fatty acids(Abercrombie K. D. 1994. Synergistic pesticidal compositions. U.S. Pat.No. 5,346,698. Mycogen Corporation. Sept. 13). Likewise, a method thatincludes concurrent administration to eliminate damages caused to plantsby nematodes, the site, soil or seeds that need treatment of a) aMyrothecium verrucaria fungus metabolite and b) a chemical pesticide, aswell as the synergistic nematicide compositions useful in this case, isclaimed under patent (Warrior P., Heiman D. F. and Rehberger Linda A.1996. Synergistic nematocidal compositions. Abbott laboratories.WO9634529, 1996-11-07).

[0005] Another approach is to combine spores of Pasteuria penetrans anematode bacterial parasite, with organophosphorated nematicides(Nordmeyer D. 1987. Synergistic nematocidal compositions of Pasteuriapenetrans spores and an organophosphorus nematocide. 1987. CIBA-GEIGY AGPatent AU 06057386A1. 01/29/1987).

[0006] However, preparation of P. penetrans spores at industrial scalefaces the problem that the organism is an obligated parasite; hence itmust be grown in in situ nematodes, isolated from nematode infested rootdigests.

[0007] Chitinolytic fungi and bacteria that share the nematode'shabitat, may have certain biological balance and somehow restrictnematode proliferation. Two strains of chitinolytic bacteria (Toda T.and Matsuda H. 1993. Antibacterial, anti-nematode and/or plant-cellactivating composition, and chitinolytic microorganisms for producingthe same. Toda Biosystem Laboratory, Japan. U.S. Pat. No. 5,208,159, May4, 1993) have been claimed as antibacterial, antinematode and/orplant-cell activating composition.

[0008] There are some examples of the chitinolytic effect on nematodes.Some of the most significant are the strains of new bacteria described(Suslow T. and Jones D. G. 1994. Novel chitinase-producing bacteria andplants. DNA Plant Technology Corporation, U.S. Pat. No. 04,940,840, Jul.10, 1990) that are created by the introduction of DNA that codifies forchitinase production, an enzyme that can degrade chitin in fungi andnematodes. The strains are useful in the production of chitinase toinhibit plant pathogens. Novel plants resistant to pathogens aredescribed too, as the result of introduction of DNA codifying forchitinase production.

[0009] Other instances of microorganisms that reduce nematodepopulations that attack plants in natural conditions are described.

[0010] Rodriguez-Kabana et al. (Rodriguez-Kabana R., Jordan J. W.,Hollis J. P. 1965. Nematodes: Biological control in rice fields-role ofhydrogen sulfide. Science. 148: 524-26); Hollis and Rodriguez-Kabana(Hollis, J. P., y R. Rodriguez-Kábana. 1966. Rapid kill of nematodes inflooded soil. Phytopathology 56, pp 1015-19) observed correspondenceamong bacterium Desulfovibrio desulfuricans, hydrogen sulfide productionand plant parasitic nematodes, whose population decreased in Louisiana'srice plantations. Sulfides are inhibitors in the electron transportbreathing process of the aerobic organism, just like other metabolitesproduced by certain soil bacteria (Rodriguez-Kábana, R. 1991. Controlbiológico de nematodes parasitos de plantas. NEMATROPICA, 21(1), pp111-22).

[0011] PAECIL™, also known as BIOACT or Nemachek, is a biologicalnematicide that contains a patented strain from Paecilomyces lilacinus,in a dry and stable spore concentration for soil and seed treatment.This fungal species is commonly found in all soils worldwide. Thepatented strain used as PAECIL™ active ingredient has a particulareffectiveness against plant parasitic nematodes. It was originallyisolated at The Philippines University, and has been developed inAustralia, Macquarie University. Furthermore, it has been broadly testedfor the control of several kinds of nematodes that attack major crops inAustralia, The Philippines, South Africa, and others. PAECIL™formulation is commercially available as a pesticide registered in ThePhilippines, under the name of BIOACT^(R); in South Africa, under thename of PL PLUS; and Indonesia, under the name of PAECIL™. Currently,the Australian National Registration Authority is evaluating the productas a pesticide (Holland, R. PAECIL™. 1998.http://www.ticorp.com.au/article1.htm). The above-mentioned instancesfail to solve all parasitic helminth problems. Therefore, the need toimplement improved means for parasite control to substitute chemicalpesticides and antiparasitic products still remains.

[0012] Trematodes cause considerable economic damage to animalproduction and human health. The diversity of the species, relativebenign pathogenicity and endemism in isolated regions seem to beessential factors that effect on the lack of knowledge on trematodes. Ingeneral terms, intestinal trematodes are zoonotic and have a largenumber of reservoir hosts in each species.

[0013] Economically speaking, one of the most significant trematodes isFasciola hepatica, the first known parasitic trematode; it affects manby inhabiting the bile conduits. Its egg is one of the largest, ovoidand operculated from helminthes, and causes digestive malfunctionconsisting in gastric disepsia, colon motility malfunction, liver andbile vesicle pain, fever and hepatic colic. Other signs may includecystic forms in lungs, eyes, brain, hepatic vein, and other tissues(Saleha A. 1991. Liver fluke disease (fasciolosis) epidemiology,economic impact and public health significance. Southeast Asian J. Trop.Med. Public health 22 supp 1dic. P 361-4)

[0014] Zoohelminths have become significant pests to sheep and cattle.Antihelminthic resistance is wide, particularly in populations of smallruminant parasitic nematodes.

[0015] New supplementary techniques have been developed, others areunder research. Fungus, Duddingtonia flagrans is a predator that formsnets, produce wide wall, motionless spores: clamidospores, able tosurvive the passage along the intestinal tract of cattle, equines, sheepand swine (Larsen M. 1999. Biological control of helminths. Int JParasitol. January; 29(1): 13946, and Larsen, M. 2000. Prospects forcontrolling animal parasitic nematodes by predacious micro fungi.Parasitology, 120, S120-S121).

[0016] Works on D. flagrans in Denmark, France, Australia, USA, andMexico, have confirmed the strong potential for biological control thisfungus has.

[0017] Like many other important sheep producing countries, South Africaundergoes a big crisis in terms of antihelminthic resistance, especiallyin gastrointestinal nematodes in sheep and goat. Significant parasitichelminthes are involved in this phenomenon; however, this causes aparticular problem with abomasum hematophage parasite Haemonchuscontortus. The studies point out that over 90% of this parasite'sstrains from the most important sheep producing regions in South Africa,show several drug resistance degrees, in three out of the fourantihelminthic groups available in the South African market. Even inareas of common grazing in Northern Province, it was detected in fiveherds studied in 1993 (van Wyk J. A., Bath G. F. and Malan F. S. 2000.The need for alternative methods to control nematode parasites ofruminant livestock in South Africa. World Animal Review.http://www.fao.org/ag/AGA/AGAP/FRG/FEEDback/War/contents.htm).

[0018] Resistance increase has become serious, since it has beenexperienced in other areas as well. A series of antihelminthic studieshave been recently conducted in four Latin American countries: Argentina(Eddi, C., Caracostantogolo, J., Peya, M., Schapiro, J., Marangunich,L., Waller, P. J. & Hansen, J. W. 1996. The prevalence of anthelminticresistance in nematode parasites of sheep in southern Latin America:Argentina. Vet. Parasitol., 62: 189-197); Brazil (Echevarria F., BorbaM. F. S., Pinheiro A. C., Wailer P. J. & Hansen J. W. 1996. Theprevalence of anthelmintic resistance in nematode parasites of sheep insouthern Latin America: Brazil. Vet. ParasitoL, 62: 199-206); Paraguay(Maciel S., Giminez A. M., Gaona, C., Waller P. J. & Hansen J. W. 1996.The prevalence of anthelmintic resistance in nematode parasites of sheepin southern Latin America: Paraguay. Vet. Parasitol., 62: 207-212); andUruguay (Nari A., Salles J., Gil A., Waller P. J. & Hansen J. W. 1996.The prevalence of anthelmintic resistance in nematode parasites of sheepin southern Latin America: Uruguay. Vet. Parasitol., 62: 213-222).

[0019] One of the nematodes that causes the greatest damages to cattleis Dictyocaulus viviparous, a parasite that comes to sexual maturity andwhen adult, is lodged in the lung of cattle, particularly young animals.The diseased caused is known as verminose bronchitis, or bovineDictyocaulosis, and infestation is produced after ingesting the 3 orinfesting larvae, present in the pastures. The treatment requiresantihelminthics (Borgsteede F. H. M, de Leeuw W. A. & Burg W. P. J.1988. A comparison of the efficacy of four different long-acting bolusesto prevent infections with Dictyocaulus viviparus in calves. TheVeterinary Quarterly, Vol 10, No. 3), but success is at the expense ofnew strains resistant to the drugs, which make further infested animaltreatment harder. The high cost of these products is a restrictivefactor to the countries with a large number of resources, and harmfulecological effects are produced with the use of these formulations.

[0020] The international problem of anthelmintic resistance iscompounded by the fact that, while chemotherapy continues to be thecornerstone of parasite control, there seems little hope that any novel,chemically unrelated anthelmintics will be forthcoming for at least thenext decade (Soll, M. D. 1997. The future of anthelmintic therapy froman industry perspective. In J. A. van Wyk & P. C. van Schalkwyk, eds.Managing anthelmintic resistance in endoparasites, p. 1-5. Proceedingsof the 16th International Conference of the World Association for theAdvancement of Veterinary Parasitology, Sun City, South Africa, Aug.10-15, 1997).

[0021] In the case of bacteria and pathogenic fungi, there arecomprehensive reports on biologicals, whose action is mainly based onantagonism and that a large amount of them are commercially available.Some of them are Conquer (Pseudomonas fluorescens that antagonizesPseudomonas tolassii), Galltrol-A (Agrobacterium radiobacter, thatcontrols Agrobacterium tumefaciens), Bio-Fungus (Trichoderma spp, thatcontrols the following fungi: Phytophthora, Rhizoctonia solani, Pythiumspp, Fusarium, Verticillium), Aspire (Candida oleophila I-182 thatcontrols Botrytis spp. and Penicillium spp), etcetera.

[0022] One of the most widely active biofungicides is Trichoderma spp(Chet I, Inbar J. 1994 Biological control of fungal pathogens. ApplBiochem Biotechnol; 48(1): 37-43) a fungus whose action mechanism islargely discussed, where chitinases that degrade the cellular wall ofthe host fungus take part. Moreover, there are experimental evidences ofchitinolytic action from fungi and bacteria used as fungal diseasebioregulators (Herrera-Estrella A, Chet 1.1999. Chitinases in biologicalcontrol. EXS; 87:171-84). However, this is not the only mode of actionof bacteria over phytopathogenic fungi; there are other control waysbased on the production of secondary metabolites, like hydrogen cyanide,that manages to inhibit root pathogenic fungi (Blumer C. and Haas D.2000. Mechanism, regulation, and ecological role of bacterial cyanidebiosynthesis. Arch Microbiol March; 173(3): 170-7), in the particularcase of P. fluorescens CHAO strain.

[0023] Analyses of bacterium-bacterium interaction have shown there arethree main types: antibiosis, substrate competition and parasitism. Inthe case of antibiosis, some bacterial strains are known to releaseantibiotics in order to suppress the surrounding bacterial activity,which may be used for biological control of pathogenic species.Likewise, substrate competition is a mechanism that may as well be usedto achieve proper biological control, since the bioregulating organismis able to synthesize siderophores microelement quelant agents, whichcauses microelement deficiency, mainly iron, in the medium, thusinhibiting the respective pathogenic growth (Ongena M. 1998. Conferenceon biological controls. Training program in the area of biotechnologyapplied to agriculture and bioindustry. Gembloux, Belgium).

DISCLOSURE OF THE INVENTION

[0024] The invention is related with a composition that contains, atleast, one chitinolytic agent or a chitinolytic activity inducing agent,and sulfide or a sulfide producing agent from microorganisms or chemicalcompounds, where the chitinolytic agent or a chitinolytic activityinducing agent, and sulfide or sulfide producing agent frommicroorganisms or chemical compounds, are concurrently applied at asubstantially minor degree than when each component is usedindependently to achieve effective control over helminths and causativeagents of bacterial and fungal diseases.

[0025] The invention is also related with the use of such compositionsand/or the concurrent administration of the said compounds fromdifferent sources, such as, biologicals and chemicals for effectivecontrol over a wide spectrum of plant parasitic nematodes (Meloidogynespp, Angina spp, Ditylenchus spp, Pratylenchus spp, Heterodera spp,Aphelenchus spp, Radopholus spp, Xiphinema spp, Rotylenchulus spp),animal parasitic nematodes and trematodes (Haemonchus spp,Trichostrongylus spp, Dictyocaulus spp. y Fasciola hepatica), bacterialagents causative of diseases (Erwinia chrysanthemi, Burkholderia glumae)and fungal agents causative of diseases (Pestalotia palmarum, Alternariatabacina, Sarocladium orizae).

[0026] The effects of a chitinolytic agent or a chitinolytic activityinducing agent and sulfide, or a sulfide-producing agent on helminths,bacteria and fungi have been previously demonstrated or reported. Inthis study, however, for the first time, a synergistic effect isdemonstrated when both components are concurrently applied.

[0027] When the chitinolytic agent, or the chitinolytic activityinducing agent and sulfide or a sulfide producing agent are separatelyapplied, the effects are always less than when the two agents aresimultaneously applied.

[0028] When applied as a composition of the present invention, thechitinolytic agent or the chitinolytic activity inducing agent andsulfide, or sulfide producing agent can be appropriately mixed in theform of a solution, suspension, emulsion, powder or granulating mixture,and is applied to the plant or soil as a fertilizer, pre-packed soil,covert seed device, a powder, granulate, nebulizer, a suspension,liquid, or any of the indicated form in capsules for the control ofparasitic helmiths, and bacterial and fungal diseases.

[0029] The optimal application ranges of the chitinolytic agent or thechitinolytic activity inducing agent and sulfide or a sulfide producingagent for the particular case of nematodes, trematodes, bacteria orfungus; and for the case of specific conditions, the ranges aredetermined through experimental studies, in vitro, greenhouse or underfield conditions.

[0030] According to the results described in the present invention, asignificant control over helminths, bacteria and fungi is achieved witha mixture of 1) a chitinase producing microorganism between 10⁷ ColonyForming Units (CFU) and 10¹² CFU of a particular microorganism percomposition gram or chitin between 1% and 50% of the composition; and 2)a sulfide producing microorganism between 10⁷ CFU and 10¹² CFU of aparticular microorganism per composition gram, or any sulfide producingchemical agent, where sulfide varies between 1.0 mg/minute percomposition gram.

[0031] Any composition with a microorganism between 10⁷ CFU and 10¹² CFUper composition gram, that concurrently produces chitinolytic agents andsulfide, is appropriate for the control over helminths, bacteria andfungi. The previous compositions involve combinations of the followingagents in the above-mentioned proportions:

[0032] 1. Chitinase and Na₂S.

[0033] 2. Chitinase and FeS.

[0034] 3. Chitinase and microorganism Desulfovibrio desulfuricans.

[0035] 4. Chitinase and Na₂S.

[0036] 5. Chitinase and FeS.

[0037] 6. Chitine and microorganism Desulfovibrio desulfuricans.

[0038] 7. Microorganism that produces chitinolytic activity and H₂Sconcurrently.

[0039] The previous compositions are effective against a wide range ofplant parasitic nematodes, including, not limiting Meloidogyne species,such as, M. incognita; Angina species, such as A. tritici; Ditylencusspecies, such as D. dipsaci; Pratylenchus species, such as P. coffee;Heterodera species, such as H. glycines; Aphelenchus species, such as A.avenae; Radopholus species, such as R. similis; Xiphinema species, suchas X. index; Rotylenchulus species, such as R. reniformis; Zoonematodessuch as: Haemonchus spp, Trichostrongylus spp, Ostertagia spp,Nematodirus spp, Cooperia spp, Ascaris spp, Bunostomum spp,Oesophagostomum spp, Chabertia spp, Trichuris spp, Strongylus spp,Trichonema spp., Dictyocaulus spp., Capillaria spp., Heterakis spp.,Toxocara spp, Ascaridia spp, Oxyuris spp, Ancylostoma spp, Uncinariaspp, Toxascaris spp and Parascaris spp; trematodes, such as Fasciolahepatica; plant pathogenic bacteria, such as Erwinia chrysanthemi,Burkholderia glumae, and plant pathogenic fungi such as Pestalotiapalmarum, Alternaria tabacina and Sarocladium orizae.

EXAMPLES Example 1 In Vitro Evaluation of the Nematicidal Effect ofHydrogen Sulfide from Chemical Sources and a Chitinolytic Enzyme.

[0040] Eggs from zoonematodes Haemonchus spp and Trichostrongyluscolubriformis and Dictyocaulus viviparus were used, as well as parasiticphytonematode larvae (juveniles 2) from Melodoigyne incognita.

[0041] Collections of Haemonchus spp and Trichostrongylus colubriformisnematodes were made from ovine (sheep) and bovine (cattle) abomasa,respectively. The adult female nematodes were washed in a physiologicalsolution and treated with “Hibitane” (Chlorhexidine Acetate) at 0.5%,for 1 minute, the process developed at 37° C. Approximately 100previously disinfected individuals were introduced into an Erlenmeyercontaining 50 ml of LB medium solution, diluted 10 times in distilledsterile water, and were left laying their eggs overnight (8-10 hours).

[0042] Collections of D. viviparous nematode were made from the infestedlung of a bovine (cattle), previously sacrificed. The same procedure wasused for Haemochus spp. and T. colubriformis; however, the females wereallowed to lay their eggs for 2-3 hours.

[0043] From that moment on, manipulation was done under asepticconditions in a vertical laminar flow, using 24-well tissue cultureplates. The total volume of the medium that contained the females andthe eggs was filtered with a sift net of 60 μm. The nematode eggs weretrapped on the 30 μm net of a second sifts. It was introduced into aHibitane solution at 0.5% for 3 minutes, followed by three washes withLB medium diluted 10 times in sterile distilled water.

[0044] Once disinfected, the eggs were removed from the sift and werecarefully resuspended with a LB medium solution diluted 10 times insterile distilled water. The final result of the distribution waschecked by counting and registering the eggs in each well with aninverted Olympus microscope, observations of the uniformity of theevolutionary state in this phase were made too.

[0045] The Haemonchus spp and T. colubfiformis' eggs hatch between 24and 48 hours of incubation at 28° C., whereas the D. vivparus' eggshatch before 24 hours. A good sample preparation is accomplished when inall the untreated controls more than 60% of hatching occurs in thepreviously foreseen times for each species.

[0046] The collection of egg mass of Meloidogyne incognita was performedfrom squash roots (Cucurbita pepo), previously infested and cultivatedin greenhouses. For this operation a stereoscope microscope and needleswith properly altered tips were used. The masses were put in steriledistilled water in Petri dishes at 28° C., in a number of 50 masses perdish. Daily observations were made to check egg hatching. Inapproximately 72 hours, there were enough larvae to start collecting anddisinfecting.

[0047] The total volume of water containing the egg masses and thelarvae were filtered through a sift net of 60 μm. From that moment onall the manipulation was done under aseptic conditions in a verticallaminar flow, using 24-well tissue culture plates. The eggs detachedfrom the mass were unable to hatch and remained on the sift net of 30μm; the larvae were collected with a further net of 5 μm. It wasintroduced into a Hibitane solution at 0.5% for 3 minutes followed by 3washes with LB medium diluted 10 times in sterile distilled water. Oncedisinfected, the Meloidogyne incognita larvae were removed from the siftnet and carefully resuspended with a LB medium solution diluted 10 timesin sterile distilled water. The final collecting and disinfectingresults were checked by counting and registering the live larvae with aninverted Olympus microscope.

[0048] The nematode's eggs and larvae were placed in a number of 100individuals in approximately 2 ml of LB medium diluted 10 times. Thisvolume was introduced into safety valves that allow the air to gothrough the liquid and, therefore, the gasses make contact with the eggsand larvae. Every valve was a replica for each treatment.

[0049] The hydrogen sulfide was obtained by a reaction against thechloride acid of two sulfide salts (Na₂S and FeS), and from ananaerobial fermentation of bacterium Desulfovibrio desulfuricans subs.desulfuricans ATCC 27774 (isolated from an ovine rumen). Thechitinolytic enzyme used was chitinase SIGMA C 1650, from bacteriumSerratia marcescens.

[0050] The nematode's eggs and larvae under the study were subjected tothe following treatments for 24 hours:

[0051] 1. Control treatment: chitinase not applied, and air circulatedthrough the valve.

[0052] 2. Chitinase treatment: chitinase at a rate of 0.2 units perreplica.

[0053] 3. Sulfide treatment: hydrogen sulfide from Na₂S with a 0.2 fluxat 0.3 mg/minute.

[0054] 4. Sulfide treatment: hydrogen sulfide from FeS with a 0.2 fluxat 0.3 mg/minute.

[0055] 5. Sulfide treatment: hydrogen sulfide from Desulfovibriodesulfuricans with a 0.2 flux at 0.3 mg/minute.

[0056] 6. Combined treatment: simultaneous application of treatments 2and 3.

[0057] 7. Combined treatment: simultaneous application of treatments 2and 4.

[0058] 8. Combined treatment: simultaneous application of treatments 2and 5.

[0059] All the above treatments had 4 replicas.

[0060] Twenty-four hours after starting the experiment the emerginglarvae (Haemonchus sp., T. colubriformis and D. viviparous) and thenumber of live larvae (Melodogyne incognita) in all the treatments, werecounted. The effectiveness results (E) are shown in table 1. This valueis the mean of the 4 replicas in every treatment. The variance analysis(ANOVA) was applied to the results obtained in each nematode species inthe study, separately; the Duncan test (Lerch G. 1977. LaExperimentación en las ciencias biológicas y agricolas. 1^(ra) edición,p.p. 203-308, Editorial Cientifico-Técnica, La Habana) was applied,which is also shown in table 1. Equal letters indicate that there are nosignificant differences (p<0.05) among the treatments. TABLE 1 Treatmenteffectiveness (E)* Treatment effectiveness (E)* 1.Ec 6. Eqsn 7. Eqsf 8.Eqsd Control 2. Eq 3. Esn 4.Esf 5. Esd (2 + 3) (2 + 4) (2 + 5)Haemonchus 0.00 0.32 0.41 0.40 0.37 0.86 0.85 0.82 (a) (b) (c) (c) (b,c) (d) (d) (d) Trichostrongilus 0.00 0.37 0.40 0.39 0.38 0.88 0.88 0.83(a₁) (b₁) (b₁, c₁) (b₁, c₁) (b₁, c₁) (d₁) (d₁) (d₁) Dictyocaulus 0.000.35 0.44 0.42 0.40 0.91 0.90 0.86 (a₂) (b₂) (c₂) (c₂) (b₂, c₂) (d₂)(d₂) (d₂) Meloidogyne 0.00 0.39 0.51 0.52 0.47 0.95 0.93 0.90 (a₃) (b₃)(c₃) (c₃) (c₃) (d₃) (d₃) (d₃)

[0061] To determine the synergic effect in treatments 6, 7 and 8, it wasassumed that the events occurring in them are not excluding.

[0062] For this type of analysis, the expected effectiveness (EE) mustbe equal to the sum of the individual effects (EI), given by theeffectiveness rendered to the chitinase action (Eq) and theeffectiveness rendered to the hydrogen sulfide action (Esn, Esf andEsd), minus the intersection effect (ei) (Sigarroa, A. 1985. Biometría ydiseño experimental. 1ra. Parte. Minist. Educación Sup. Ed. Pueblo yEducación. Cap. 3. pag 69-107).

EE=Eq+Es−ei, where ei=Eq×Es

[0063] If the experimental effectiveness (E) in the treatments where twonematicidal agents combine (treatments 6, 7, 8) is greater than theexpected effectiveness (EE) for those treatments, it can be assured thatthere is synergism in terms of the nematicidal activity of thechitinolytic agent (chitinase) and the hydrogen sulfide when both areconcurrently applied in the same treatment. The values obtained aresummarized in table 2. TABLE 2 Experimental (E) and expected (EE)effectiveness. Experimental (E) and expected (EE) effectiveness.Tratamiento 6 Tratamiento 7 Tratamiento 8 E EE E EE E EE Haemonchus 0.860.60 0.85 0.59 0.82 0.57 Trichostrongilus 0.88 0.62 0.88 0.62 0.83 0.61Dictyocaulus 0.91 0.64 0.90 0.62 0.86 0.61 Meloidogyne 0.95 0.70 0.930.71 0.90 0.68

[0064] In the three treatments where chitinase and hydrogen sulfide aresimultaneously combined, the experimental effectiveness (E) was greaterthan the expected effectiveness (EE) for the four nematodes under thestudy, which statistically demonstrates the existence of synergismbetween both compounds (when they act concurrently), regarding theirnematicidal activity. No significant differences were observed as to theorigin of the sulfides and their nematicidal effect (TABLE1).

Example 2 Greenhouse Evaluation of the Nematicidal Effect of aChitinolytic-Activity Inducing Agent (Chitin) and a HydrogenSulfide-Producing Agent (Desulfovibrio desulfuricans subps.desulfuricans ATCC 29577 Isolated from the Soil).

[0065] Brown soil with neutral pH was selected: it was dried and sievedwith a 0.5 cm net to remove the undesirable particles. It was sterilizedin a vertical autoclave for 1 hour at 120° C. and 1 atmosphere (SambrookJ., Fritsch E. F. and Maniatis T. 1989. Molecular Cloning: A LaboratoryManual. 2nd. Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., USA). It was dried at room temperature for 3-4 days to later makethe foreseen mixtures in the treatments with river sand, soil worm humusand chitin (ICN catalogue number 101334).

[0066] Twenty pots (15 cm diameter×13 cm depth and 1 liter of capacity)were filled with the set proportions in the following treatments:

[0067] 1. Control treatment: soil 70%, river sand 25% and humus 5%.

[0068] 2. Chitin treatment: soil 70%, river sand 25%, humus 4% andchitin 1%.

[0069] 3. Microorganic treatment: soil 70%, river sand 25%, humus 5% andD. desulfuricans, applied to a concentration of 1010 CFU-pot.

[0070] 4. Combined treatment: soil 70%, river sand 25%, humus 4%, chitin1% and D. desulfuricans applied to a concentration of 1010 CFU/pot.

[0071] Each treatment was carried out with 5 replicas (pots).

[0072] In treatments 2 and 4 a pre-mixture of humus with chitin was madein a 4:1 proportion, followed by a final mixture with the soil and thesand. In treatments 3 and 4, D. desulfuricans was applied with 100 ml ofde-ionized water per pot. These volumes were uniformly applied duringthe first irrigation.

[0073] For all the treatments, 500 nematode specimens of Radopholussimilis previously collected from naturally infested banana roots wereinoculated in the pots. The centrifugation-floatation technique(Jenkins, W. R. 1964. A rapid centrifugal-flotation tecnique forseparation nematodes from soil. Plant Disease Reporter, 48: 692) wasused; the specimens were diluted in 5 ml of distilled water anduniformly applied at a depth of 5 cm under the soil surface.

[0074] The pots were placed in greenhouses and remained still for threedays after applying the treatments and inoculating the nematodes. Dailyirrigation was performed during this stage, in order to preserve thegood moisture conditions. Before the fourth day of treatments, a bananaplant var. Cavendish, achieved by in vitro tissue culture, wastransplanted to the pots. From that moment on a strict irrigation regimefollowed, which allowed permanent soil moisture in its field capacity.

[0075] The final evaluation was done three months after the experimentwas initiated, the plant's roots were carefully removed from the soil.Then the number of specimens (larvae and adults) and live nematodescollected from the plants, were registered, using thecentrifugation-floatation technique (Jenkins, W. R. 1964. A rapidcentrifugal-flotation tecnique for separation nematodes from soil. PlantDisease Reporter, 48: 692), and an inverted microscope for the counts.The effectiveness results for the different treatments are shown intable 3. This is the mean value of the 5 replicas for each treatment.The variance analysis was applied to the results achieved (ANOVA),followed by the Duncan test (Lerch G. 1977. La Experimentación en lasciencias bioló-gicas y agricolas. 1^(ra) edición, p.p. 203-308,Editorial Cientifico-Técnica, La Habana), shown in table 3. Equalletters indicate that that there are no significant differences (p<0.05)among the treatments. TABLE 3 Treatment effectiveness (E)* Treatmenteffectiveness (E)* 1. Ec 2. Eq 3. Esd 4. Eqsd Radopholus similis 0.00(a) 0.21 (b) 0.18 (b) 0.48 (c)

[0076] To determine the possible synergic effect in treatment 4, it wasassumed that the occurring events (nematicidal effect), are notexcluding.

[0077] Like Example 1, the expected effectiveness (EE) must be equal tothe sum of the individual effects (EI), given by the effectivenessrendered to chitin action (Eq) as an inductor of the chitinolyticactivity of the microorganisms present in the mixture of soil and humus,and the effectiveness rendered to the action of hydrogen sulfide (Esd)from bacteria D. desulfuricans; minus the intersection effect (ei)between the two treatments (Sigarroa, A. 1985. Biometria y diseñoexperimental. 1ra. Parte. Minist. Educación Sup. Ed. Pueblo y Educación.Cap. 3. pag 69-107)

EE=Eq+Es−ei, where ei=Eq×Es

[0078] If the experimental effectiveness (E) in treatment 4 where thetwo nematicidal agents are combined, is greater than the expectedeffectiveness (EE), it can be assured that there is synergism betweenthe chitinolytic activity-inducing agent (chitin) and hydrogen sulfide(from D. desulfuricans), where they are concurrently applied in the sametreatment. The values obtained are shown in table 4. TABLE 4Experimental (E) and expected (EE) effectiveness. Experimental (E) andexpected (EE) effectiveness Treatment 4. E EE Radopholus similis 0.480.35

[0079] In treatment 4 a chitinolytic activity inductor (chitin), and abiological source of hydrogen sulfide (D. sulfuricans) are combined. Inthis case the experimental effectiveness (E) was greater than theexpected effectiveness (EE), thus proving the existence of synergism(regarding its nematicidal activity) in the two compounds when they areconcurrently applied in the soil.

Example 3 Demonstration of Chitinolytic Activity and Sulfide Productionfrom Bacteria Corynebacterium paurometabolum C-924 and Tsukamurellapaurometabola DSM 20162.

[0080] Sulfide Production Determination:

[0081] In tubes of 100 ml for gas collection, samples from the gascurrent from fermentation of strains C-924 and DSM 20162 in 51bioreactors, were taken. The total culture time was 24 h. The formationof hydrogen sulfide was detected first at the 16^(th) h.

[0082] The samples were processed in an analogous manner to the H₂Spattern generated. The analysis was performed in the Varian gaschromatograph, following these conditions:

[0083] Flame photometric detector with filter sensitive to compoundsthat contain sulfur.

[0084] Hydrogen sulfide pattern: 43.2 ng/ml, by duplicate.

[0085] Samples: duplicate for each time when sampling was done.

[0086] Injection: 1 ml or μl of head space.

[0087] Column: DB-5 (15 m×0.53 mm)

[0088] Temperature: 35° C.

[0089] Carrier gas: Nitrogen 1.5 ml/min.

[0090] Detector: FPD-S

[0091] Purge gas: Nitrogen 30 ml/min.

[0092] Table 5 shows a summary of the sulfide gases analysis issued bythe two strains at different times. TABLE 5 Sulfide gases analysis H₂Sflux mg/min (Sulfide flux detected) 18 24 Strains Samples 16 hours hours20 hours 22 hours hours C-924 1 0.0673 0.2208 0.4779 0.3578 0.0672 20.0659 0.2160 0.4755 0.3552 0.0680 DSM 1 0.0231 0.0416 0.1014 0.18630.0009 20162 2 0.0240 0.0422 0.1040 0.1887 0.0097

[0093] Both strains produce sulfides, but C-924 produces higher fluxthan strain DSM 20162.

[0094] Chitinolytic Activity Determination:

[0095]Corynebacterium paurometabolum C-924, Tsukamurella paurometabolaDSM 20162, Serratia marcescen ATCC 13880 and E. coli ATCC 25922 strains,were used.

[0096] The bacterial cultures of the studied strains were grown in LBmedium at 28° C. and 100 rpm for 24 hours, followed by centrifugation at3500 rpm; the supernatants were filtered through two 0.2 μm nets. Thefiltered product was assayed in plates prepared with a chitin colloidalsuspension (0.5%), agarose was added too, up to 0.8%, to achieve themedium gelling and assure porosity to facilitate protein diffusion.After gelling, 5 mm diameter wells were opened, where 100 μl of thefiltered supernatant from each bacterial strain was added. Threereplicas were used for every plate, and were incubated at 28° C. in thedark.

[0097] From the 48^(th) hour on, a decrease was observed in the mediumturbidity resembling a halo, which demonstrated chitin hydrolysis. Inthe following table (TABLE 6), the qualitative results from theoccurrence of a hydrolysis halo at different incubation times with thesupernatant from the culture of the different strains studied, areshown. TABLE 6 Occurrence of a hydrolysis halo. Strains 24 hours 48hours 72 hours S. marcescen. Negative Positive + Positive + + + C.paurometabolum Negative Negative Positive + + T. paurometabola. NegativeNegative Positive + E. coli. Negative Negative Negative

[0098] Both strains (C. paurometabolum and T. paurometabola) showed thechitin-hydrolysis halo, just like the positive control used (S.marcescen), whereas the E. coli strain (negative control) did notproduce a hydrolysis halo.

Example 4 In Vitro Evaluation of Effects from Sulfides and ChitinasesProduced by Bacteria Corynebacterium paurometabolum C-924 andTsukamurella paurometabola DSM 20162, on Parasite Fasciola hepatica(Trematode).

[0099] Eggs from parasite Fasciola hepatica were used. The eggcollections were directly made from the infested liver bile of a bovine(cattle), previously sacrificed. The bile content was resuspended in a 3times higher volume of distilled water and remained still for 2-3 hoursat 28° C., to achieve egg precipitation. Then the greatest possiblevolume of supernatant liquid was removed. The precipitate was filteredthrough a sift net of 71 μm, where the eggs were trapped.

[0100] From that moment on, all the manipulation was done under asepticconditions in a vertical laminar flow, using 24-well tissue cultureplates. The sift with the F. hepatica eggs was introduced into aHibitane solution at 0.5% for 3 minutes, followed by 3 washes with LBmedium diluted 10 times in sterile distilled water. Once disinfected,the eggs were removed from the sift and were carefully resuspended witha LB medium solution diluted 10 times in sterile distilled water. Thefinal collecting and disinfecting results were checked by counting andregistering the live larvae with an inverted Olympus microscope.Observations regarding the uniformity of the evolutionary state in thisphase, were made as well.

[0101] This parasitic trematode's eggs hatch under the previously invitro set conditions in about 15 days of incubation at 28° C.; a goodpreparation of the sample was considered when more than 60% of the eggshatched at the end of the incubation period.

[0102] To develop the experiment, the disinfected eggs were placed in anumber of 100 individuals approximately, in 1 ml of LB medium diluted 10times. The volume was uniformly introduced in 20 safety valves thatallow the air passage through the liquid; hence, the gases make contactwith the eggs. Each valve was a replica (4 per treatment) in all thefive treatments.

[0103] The F. hepatica eggs were exposed to the following treatmentsduring the last 4 days of incubation:

[0104] 1. Control treatment: Addition of 1 ml of LB medium diluted 10times to every valve, with no chitinase, and air circulating through it.

[0105] 2. Addition to each valve of 1 ml of a chitinolytic supernatantwithout bacterial cells from a culture of 1010 Colony Forming Units permilliliter (CFU/ml) of Corynebacterium paurometabolum C-924.

[0106] 3. Addition to each valve of 1 ml of a chitinolytic supernatantwithout bacterial cells, from a 1010 CFU/ml of Tsukamurellapaurometabola DSM 20162.

[0107] 4. The flux of gases from a continuous culture of Corynebacteriumpaurometabolum C-924 at 1010 CFU/ml, was allowed to go through thevalves.

[0108] 5. The flux of gases from a continuous culture of Tsukamurellapaurometabola DSM 20162 at 10¹⁰ CFU/ml, was allowed to go through thevalves.

[0109] 6. Combined treatment: simultaneous application of treatments 2and 4.

[0110] 7. Simultaneous treatment: simultaneous application of treatments3 and 5.

[0111] On the fourth day following the start of the experiment, thehatched eggs were counted. In the case of F. hepatica, it was notpossible to count the larvae (miracides) that come out due to the greatmotility they have; therefore, observations through the microscope arefocused on the eggs. The effectiveness results from the differenttreatments are shown in table 7. This is the mean value for the 4replicas in each treatment. Equal letters indicate the lack ofsignificant differences (p<0.05) among the treatments. TABLE 7 Treatmenteffectiveness (E)* Treatment effectiveness (E)* 1. E 2. Eq 3. Eq 4. Es5. Es 6. E 7. E Control C-924 DSM20162 C-924 DSM20162 (2 + 4) (3 + 5)Fasciola 0.00 0.18 0.11 0.29 0.16 0.52 0.28 hepatica (a) (b) (c) (d)(b,c) (e) (d)

[0112] To determine the possible synergic effect in treatments 6 and 7,it was assumed that the events (anti-parasitic effect) occurring inthem, are not excluding.

[0113] For this type of analysis, the expected effectiveness (EE) isgiven by the effectiveness rendered to the chitinase action (Eq) and theeffectiveness rendered to the action of hydrogen sulfide (Esn, Esf andEsd), minus the intersection effect (ei) (Sigarroa, A. 1985. Biometria ydiseño experimental. 1ra. Parte. Minist. Educación Sup. Ed. Pueblo yEducación. Cap. 3. pag 69-107).

EE=Eq+Es−ei, where ei=Eq×Es

[0114] If the experimental effectiveness (E) in the treatments where twoanti-parasitic agents combine (treatments 6 and 7), is greater than theexpected effectiveness for these treatments, it can be assured thatthere is synergism in terms of the anti-parasitic activity of thechitinolytic agent (chitinase) and hydrogen sulfide when both areconcurrently applied in the same treatment.

[0115] The values obtained are summarized in table 8. TABLE 8Experimental (E) and Expected (EE) effectiveness. Experimental (E) andExpected (EE) effectiveness. Treatment 6 Treatment 7 E EE E EE Fasciolahepatica 0.52 0.31 0.28 0.25

[0116] In the treatments where chitinase and hydrogen sulfide arecombined, the experimental effectiveness (E) was greater than theexpected effectiveness (EE), which demonstrates the synergism of the twocompounds when acting concurrently in terms of their nematicidalactivity.

Example 5 In Vitro Effect Evaluation of a Bacterial Strain(Corynebacterium paurometabolum C-924) Which Produces Hydrogen Sulfideand has Chitinolytic Ativity on Aeveral Bacteria and Fungi.

[0117] The following fungus species were used: Pestalotia palmarum,Alternaria tabacina, Sarocladium orizae, Pitium debaryanum; and thefollowing bacterial species: Erwinia chrysanthemi, Burkholderia glumae,Serratia marcescen ATCC 13880, Bacillus subtilis F 1695 and Escherichiacoli ATCC 25922, were used as well.

[0118] A) Fungus Assay.

[0119] The interaction of Corynebacterium paurometabolum C-924 on fungiwas assayed on these fungi: Pestalotia palmarum, Alternaria tabacina,Sarocladium orizae and Pytium debayianum. Strain of Serratia marcescenATCC 13880 was used as the positive control for fungicidal activity andE. coli strain ATCC 25922 was used as the negative control forfungicidal activity. The bacterial cultures were grown with the usualshaking and temperature conditions for all species in 24 hours. Thenecessary dilutions were made with absorbance at A 530 nm to assure acell concentration of 10⁹ cfu/ml. They were placed in petry dishescontaining PDA medium (agar-potato-dextrose), the inocula were made witha central line and the aid of the microbiological loop. The dishes wereincubated for 48 hours at 28° C., then the 8 mm diameter discs from thedifferent fungal strains previously grown were inoculated (platescontaining PDA medium) and placed on the plate's surface at either poleregarding the central line of the inoculated bacteria. Three replicaswere used for each fungus to be studied and were incubated for 10 daysat 28° C. The results were read from the fifth day of the beginning ofthe experiment on.

[0120] b) Bacterium Assay.

[0121] The incidence of the interaction of Corynebacteriumpaurometabolum C-924, E. coli ATCC 25922 and Bacillus subtilis F 1695was studied in these bacteria: Erwinia chrysanthanem and Burkholderiaglumae. The Bacillus subtilis strain F 1695 was used as the positivecontrol for antagonism with other bacteria, for the negative control E.coli strain ATCC 25922 was used. The bacterial strains were grown in LBmedium under the usual shaking and temperature conditions for 24 hours.From these cultures, the necessary dilutions were made, with a previousabsorbance reading at A 530 nm to assure a cell concentration of 10⁹cfu/ml. In the case of C-924, drops of 5 μl were applied on threedifferent sites on plates with LB medium, on two different sites for thepositive control and two other different sites for the negative control,respectively. The plates were incubated at 28° C. for 48 hours. Afterthat time they were treated with chloroform steam for 3 minutes (toinactivate and avoid dispersion in further steps), then the plates wereleft in the laminar flow, half-open, to eliminate the gas excess.Inoculation of the challenging strains Erwinia chrysanthemi andBurkholderia glumae, was carried out, which started with pure culturesfrom every microorganism from which the necessary amounts to make acellular concentration of 10⁹ cfu/ml were taken, after adding up tothree milliliters of semi-solid LB medium (0.1% technical agar No. 3)The mixture was dispersed on the plates containing the challengedstrains, then they were incubated at 28° C. for 48 hours to evaluate theresults.

[0122] Table 9 shows the description of the results accomplished duringthe above mentioned interaction assays. TABLE 9 Results accomplishedduring interaction assays. Antagonistic effect of Species Descriptionstrain C-924. Pestalotia palmarum Fungus, Deuteromiceto, + + +phytopathogenic of foliage and fruits. Alternaria tabacina Fungus,Deuteromiceto, + + + phytopathogenic of tobacco leaves. Sarocladiumorizae Fungus, Deuteromiceto, + + phytopathogenic of rice, it isinvolved in the acarus-fungus complex, affecting seeds, sheath and neck.Pytium debaryanum Fungus, Oomiceto, lives on the soil + and is part ofthe causative Damping-off complex. Erwinia Bacterium, isolation ofDahlia + + + chrysanthemi stems with soft rottenness symptoms.Burkholderia glumae Bacterium, isolation of rice plants + + with apicaland marginal necrosis. Bacillus subtilis Bacterium, isolation of potato− cepa F1695 rhyzosphere in rottenness-free on affected field.Biorregulator.

[0123] As shown in table 9, there is a marked antagonist effect ofstrain Corynebacterium paurometabolum C-924 on fungi Pestlotioapalmarum, Altemaria tabacina and Sarocladium orizae, which arecharacterized by having a high chitin content in their structures. Onlya slight antagonism caused by the action of hydrogen sulfide wasobserved. In the case of the interaction with the bacteria studied, theantagonism was observed in the two pathogenic strains (Erwiniacrhysanthemi and Burkholderia glumae), whereas antagonism was notobserved in the case of Bacillus subtilis, as it is isolated from anantagonist soil with other microorganisms and; therefore, more resistantto adverse environmental factors.

1. A composition for agricultural and veterinary use that comprises atleast one chitinolytic agent or a chitinolytic activity-inducing agent,and sulfide or a sulfide-producing agent.
 2. A composition of claim 1,wherein said chitinolytic agent is a chitinase.
 3. A composition ofclaim 1 wherein said chitinolytic agent is a chitinase-producing agent.4. A composition of claim 3, which contains between 10⁷ Colony FormingUnits (CFU) and 10¹² CFU of the said microorganism per composition gram.5. A composition of claim 1, wherein said chitinolytic activity-inducingagent is chitin.
 6. A composition of claim 5, wherein chitin rangesbetween 1 and 50%.
 7. A composition of claim 1, wherein saidsulfide-producing agent is a microorganism.
 8. A composition of claim 7,which contains between 10⁷ CFU and 1012 CFU of the said microorganismper composition gram.
 9. A composition of claim 1, wherein saidsulfide-producing agents are chemical agents.
 10. A composition of claim1, wherein the sulfide production ranges from 0.1 mg/minute to 1.0mg/minute per composition gram.
 11. A composition of claim 1, whereinthe chitinolytic agent and the sulfide-producing agent are amicroorganism.
 12. A composition according with claims 1 to 11, for thecontrol of parasitic zoonematodes.
 13. A composition according to claim12, wherein parasitic zoonematodes are Haemonchus spp., Trichostrongylusspp and Dictiocalus spp.
 14. A composition according with claims 1 to 11for the control of plant parasitic nematodes.
 15. A compositionaccording with claim 14, wherein the plant parasitic nematodes areMeloidogyne spp and Radopholus similis.
 16. A composition according withclaims 1 to 11, for the control of plant and animal pathogenic bacteria.17. A composition according with claim 16, wherein the pathogenicbacteria are, Erwinia spp. and Burkholderia spp.
 18. A compositionaccording with claims from 1 to 11 for the control of plant and animalpathogenic fungi.
 19. A composition according with claim 18, wherein thepathogenic fungi are Pestalotia palmarum, Altemaria tabacina andSarocladium orizae.
 20. A composition according with claims from 1 to11, for the control of parasitic trematodes.
 21. A composition accordingwith claim 20, wherein the parasitic trenematode is Fasciola hepatica.22. A composition for agricultural and/or veterinarian use according tothe previous claims, wherein said proper carrier such as, a fertilizer,a pre-packed soil, a seed covering device, a powder, a granulate, anebulizer, a suspension, a liquid, or any of the forms indicated, in anencapsulated formulation for the control of parasitic helminthes,bacteria and pathogenic fungi.